Reactive oxygen species [“ROS”] are chemically reactive molecules containing oxygen and comprising unpaired valence shell electrons. ROS, such as hydroxyl radicals, superoxide anions, and hydrogen peroxide [“H2O2”], are generated continually as by-products of aerobic metabolism in cells, e.g., via incomplete reduction of oxygen to water during respiration. ROS are also produced during beta-oxidation of fatty acids by exposure to radiation, light, metals, and redox active drugs. Since ROS may perturbate the cellular redox status and ultimately cause toxic damage to cellular components, including lipids, proteins, and DNA, cells must possess a variety of means to sense levels of ROS and transduce the signal such that the cell is protected against the effects of oxidative stress and cellular integrity is maintained.
Typically, levels of ROS are controlled by use of the glutathione reduction-oxidation (re-dox) cycle and thioredoxin system, such that electrons are accepted from NADPH and utilized to reduce H2O2 to water. More specifically, the electrons are transferred from NADPH to thioredoxin reductase to thioredoxin to peroxiredoxins to H2O2, yielding water. Regulation of the multiple genes in this pathway is complex. The adaptive response to H2O2 in the yeast Saccharomyces cerevisiae has been found to involve a change in the expression of at least 167 proteins (Godon, C. et al., J. Biol. Chem., 273:22480-22489 (1998)).
One means to sense levels of H2O2 in the yeast S. cerevisiae relies on a signaling pathway based on the master transcription factor for the oxidative stress response, i.e., the transcription factor protein Yap1. In response to H2O2 stress, a multi-step conformational change in Yap1 occurs based on the formation of at least one intra-molecular disulfide bond, a reaction catalyzed by peroxiredoxins such as Tsa1 and Gpx3 and facilitated by other proteins such as Ybp1. In this active oxidized form, Yap1 controls the expression of a large regulon of at least 32 different proteins, including those involved in cellular antioxidant defenses and glutathione/NADPH regeneration (Lee, J. et al., J. Biol. Chem., 274:16040-16046 (1999)). Deactivation of Yap1 occurs by enzymatic reduction with Yap1-controlled thioredoxins, thus providing a mechanism for autoregulation. Mutant strains of S. cerevisiae lacking a functional Yap1 protein are hypersensitive to killing by H2O2.
It is known that fatty acids having more double bonds are more susceptible to lipid peroxidation. Thus, polyunsaturated fatty acids [“PUFAs”] are more susceptible to oxidative degradation by ROS because they contain multiple double bonds in between which lie methylene-CH2-groups that possess especially reactive hydrogens. Avery, A. M. and S. V. Avery (J. Biol. Chem., 276:33730-33735 (2001)) reported that a S. cerevisiae gpx1Δ/gpx2Δ/gpx3Δ mutant was defective for growth in medium supplemented with the PUFA alpha-linolenic acid [“ALA”; 18:3], wherein ALA can comprise up to 60% of the total membrane fatty acids; gpx1Δ, gpx2Δ and gpx3Δ mutants also demonstrated toxicity to the 18:3, although the effect was delayed based on the slower incorporation rate of exogenous 18:3 into membrane lipids.
Since ROS are continually produced in cells performing aerobic metabolism and since ROS can lead to cell damage and death, one of skill in the art will appreciate methods that increase the capacity of recombinantly engineered organisms to defend against ROS. This is especially true in those organisms that produce microbial oils, since the generation of ROS in certain microbial strains during production of these oils can lead to lower yields and/or reduced efficiency in microbial oil production.
It has been found that engineering oleaginous yeast to have increased Yap1 transcription factor activity and to produce PUFAs results in both increased lipid content [“TFAs % DCW”] and increased average PUFA titer [“PUFA % DCW”].